Improved Object Recognition Memory Using Post-encoding Repetitive Transcranial Magnetic Stimulation Part 2
Aug 16, 2024
3. Results
We used the ORT with a 72-h delay between the sampling and test phase to assess the effectiveness of rTMS on memory. There was no difference in the OFT activity and object exploration times in the sampling and test trial between SHAM v TMS (p > 0.05, Fig. 1BD).
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Additionally, there was no bias for object location in the acquisition trial (p > 0.05, data not shown). The mice that received the TMS over 72-h had a significant discrimination index of the novel object (One-way t-test t(9) ¼ 3.067, p ¼ 0.0134), whereas SHAM mice did not (One-way t-test t(10) ¼ 0.705, p ¼ 0.496).
Comparing the two discrimination indices, TMS was significantly greater than the SHAM (Student t-test t(19) ¼ 2.77, p ¼ 0.0122, Fig. 1E). In addition, only the group that received rTMS had a significant t- We also analyzed the difference within each treatment group of exploration of the novel and familiar object and found a significant difference between the object explored and the treatment given (Two Way Repeated Measures ANOVA F(1, 19) ¼ 8.305, p ¼ 0.0096, hp 2¼.304 Fig. 1F).
Post-hoc analysis showed that the TMS group had a significant increase in novel object exploration (p ¼ 0.0092), but no difference in the SHAM group (p > 0.05), indicating that rTMS between the learning and retrieval event better consolidated the memory.
Following this result, we were interested in understanding the neurobiological changes associated with improved memory performance in mice that received rTMS. We used Western Blot to analyze the amount of CREB, CAMKII, and ERK and their phosphorylation using the Full homogenate from the hippocampus and FC (Fig. 2).
In the hippocampus, overall, there was a change in the amount of CREB (One-Way ANOVA F(2,15) ¼ 6.20 p ¼ 0.013, hp 2¼.488 Fig. 2a). Post-hoc analysis revealed that this was derived from an increase in CREB in both the groups which did the behavioral test, SHAM (p ¼ 0.048) and TMS (p ¼ 0.016) compared to the CONTROL group.
However, it was the phosphorylation of CREB that appeared to be affected solely by the rTMS in the hippocampus (One-Way ANOVA F(2,15) ¼ 11.813 p ¼ 0.001, hp 2¼.645 Fig. 2a), as post-hoc analysis showed the mice that received rTMS had significantly greater levels of pCREB than the CONTROL (p ¼ 0.001) and the SHAM group (p ¼ 0.041).
Additionally, we found an effect on the levels of pCAMKII (One-Way ANOVA F(2,15) ¼ 4.817 p ¼ 0.029, hp 2 ¼.445, Fig. 2d), where similarly to pCREB post-hoc testing showed that rTMS increased the amount of pCAMKII compared to the CONTROL group (p ¼ 0.027). In the frontal cortex, the phosphorylation of CREB appeared to be affected by the rTMS (One-Way ANOVA F(2,15) ¼ 10.572 p ¼ 0.003, hp 2¼.658, Fig. 2b).
Post-hoc analysis showed the mice that received rTMS had significantly greater levels of pCREB than the CONTROL group (p ¼ 0.002)/There was also an effect on pCAMKII in the FC (One-Way ANOVA F(2,15) ¼ 6.452 p ¼ 0.011, hp 2¼.498, Fig. 2e), however posthoc analysis showed that it was regardless of the treatment as both SHAM (p ¼ 0.016) and TMS (0.035) groups had increased pCAMKII compared to the CONTROL group.
In the FC the increased pCAMKII was due to increased phosphorylation of existing CAMKII, as the proportion of pCAMKII to CAMKII was also increased with the behavioral test (One-Way ANOVA F(2,15) ¼ 7.387 p ¼ 0.007, hp 2¼.532, Fig. 2e). Post-hoc testing showed that both SHAM (p ¼ 0.026) and TMS (0.010) had a significantly greater portion than CONTROL. In both the hippocampus and FC we did not see any significant changes in ERK or its phosphorylation after rTMS.
We used Western Blot on the Synaptic homogenates from the hippocampus and FC to determine the effect of rTMS on different synaptic receptors involved in learning and memory.
We examined the change in AMPA receptor subunits GluR1 and GluR2, as well as the BDNF receptor TrkB. In the hippocampus there was a difference in the levels of GluR2 (Welch Test F(2,15) ¼ 6.718 p < 0.001, Fig. 3g) and pGluR2 (One-Way ANOVA F(2,15) ¼ 7.189 p ¼ 0.023, hp 2 ¼.461, Fig. 3g).
Post-hoc analysis revealed SHAM mice had less GLUR2 than the CONTROLS (Games-Howell p ¼ 0.022) and the mice that received rTMS treatment had significantly higher levels of both GluR2 (Games-Howell p ¼ 0.037) and pGluR2 (p < 0.05) than the SHAM mice. In the hippocampus, we also found changes to pTrKB (One-Way ANOVA F(2,15) ¼ 8.449 p ¼ 0.005, hp 2¼.585, Fig. 3J).
As post-hoc testing showed both the SHAM mice (p ¼ 0.008) and TMS mice (p ¼ 0.013) had less pTRKb than CONTROL it suggests this effect was related to the behavior test and independent of treatment.
This effect was also due to a change in the proportion of total TrkB that was phosphorylated (One-Way ANOVA F(2,15) ¼ 8.882 p ¼ 0.004, hp 2¼.597, Fig. 3J), both SHAM (p ¼ 0.042) and TMS (p ¼ 0.004) were significantly lower than CONTROL.
The only effect on GluR1 was found in the FC where there was a change in the proportion of GluR1 phosphorylated at the S845 site,pGluR1(845) (One-Way ANOVA F(2,15) ¼ 4.170 p ¼ 0.040, hp 2 ¼.391, Fig. 3e), but not the S831 site.
Post-hoc analysis showed that there was a decrease after rTMS compared to CONTROLS (p ¼ 0.038). There was no change in GluR2 receptors in the FC however, we found an effect on the levels of TrkB (One-Way ANOVA F(2,15) ¼ 13.601 p ¼ 0.001, hp 2¼.712, Fig. 3K) and pTrkB (Welch Test F(2,15) ¼ 4.745 p ¼ 0.035, Fig. 3K).
Post-hoc analysis showed that this effect was only due to an increase in levels after rTMS treatment compared to controls for both TrkB (p ¼ 0.001) and pTrKB (Games-Howell p ¼ 0.037).
As there was no difference in the proportion of pTrKb to TrkB (Fig. 3K) this suggests that unlike in the hippocampus, the change in pTrkB was a result of the overall increase in TrKB.
Finally, we investigated the effect of rTMS stimulation on increasing and maintaining synaptic connections in areas of the brain that are involved in the ORT. We used immunohistochemistry to determine colocalized presynaptic (SV2A) and postsynaptic (PSD95) puncta to indicate the proportion of active, connected synapses in these areas.
Overall, there was change in the number of colocalized synapses in the CA1 region of the hippocampus (OneWay ANOVA F(2,16) ¼ 4.231 p ¼ 0.038, hp 2 ¼.377, Fig. 4a), the EC (One-Way ANOVA F(2,16) ¼ 4.658 p ¼ 0.030, hp 2¼.392, Fig. 4c) and the PC (One-Way ANOVA F(2,16) ¼ 7.895, p ¼ 0.006, hp 2 ¼.548, Fig. 4d).
In both the CA1 region and PC there was increased colocalization in the TMS group to CONTROL (CA1 p ¼ 0.031, PC p ¼ 0.005), however in the EC there was an increase from CONTROL in both the SHAM (p ¼ 0.047), and TMS (p ¼ 0.022) groups, suggesting an overall effect of the behavior test in this region with little effect of the treatment. We did not see any significant changes in the FC.
4. Discussion
Our results show that rTMS if given between encoding and retrieval, improves memory in the ORT in mice. These results reflect human studies which have shown a similar effect of post-encoding rTMS or other forms of non-invasive brain stimulation, on improving memory [2,3].
Predominantly rodent studies have focused on pre-encoding rTMS for improving memory in the past [29e32], therefore the neurobiological mechanisms of consolidation targeted rTMS, even though for specific memory improvement it is more effective than other stimulation time points [1], have not been investigated.
An indicator of persistent LTM is the formation of strong, stable synaptic connections. In both the PC and CA1 only the mice which had rTMS had increased synaptic connections compared to the CONTROL mice, whilst SHAM mice did not.
The PC and dorsal CA1 are important in object recognition memory through distinct mechanisms [33]. While the PC is involved in the recognition of objects based on their novelty or familiarity, the hippocampus is involved in encoding the specific object information from sensory stimuli into memory [33e35]. As both regions had elevated synaptic connections in the rTMS mice, which had improved memory, this suggests that the stimulation was maintaining both of the important connections in these areas that encode both the object information and familiarity.
One of the most striking biological changes we observed was the increase in the expression of GluR2 at the synapse of the hippocampus after rTMS treatment improved memory compared to a decrease in the SHAM mice.
This was in contrast with very little change to GluR1 expression. Previous studies have shown that GluR2 receptors are important for LTM storage and the removal of GluR2 receptors from the synapse is involved in forgetting [36]. Contrarily, blocking GluR2 endocytosis prevents forgetting in experimental settings [37].
There have only been a few studies that have shown conflicting evidence of the effect of rTMS on GluR2 receptors [26,38,39]. We believe our results show a unique effect as the rTMS was paired with the learning task which resulted in the forgetting and loss of GluR2 receptors in the SHAM mice.
We therefore have shown for the first time that rTMS can promote the stability of GluR2 receptors at the synapse about the object recognition task, which as demonstrated in previous literature, is important to maintain a memory [36,37].
Interestingly in the FC, rTMS was associated with an increase in TrkB and the phosphorylation of TrKB, whereas in the hippocampus, the memory task reduced pTrkB expression. The kinetics of TrkB at the synapse are dependent on many factors, as TrkB can be internalized at the synapse once BDNF binds [40].
However, longer activation periods of BDNF result in a stable upregulation of TrkB whereas transient BDNF increases TrkB only temporarily [41]. Therefore, the differing expression of FC and hippocampus TrkB may be an indication of the region-specific effects of BDNF.
In the hippocampus, TrkB internalization may have occurred in both the SHAM and TMS mice in response to the behavioral test, as conducting the test phase may have caused some level of reconsolidation learning event in both of these groups, which would have activated a short BDNF response [42e44]. Whereas in the FC, as previous studies have shown direct increases in BDNF from rTMS [18e20], the persistent increase in BDNF from the stimulation over the three days may have promoted the upregulation and translocation of TrkB to the synapse [41].
Additionally, the effect of the behavior test causing TrkB internalization would not have occurred in the FC as it did in the hippocampus, as the FC is not involved in recognition memory [45e47]. This was also demonstrated in our study as there was no change in synaptic connections in this region. Future studies should explore rTMS versus behavioral test effects in more detail to determine this exact mechanism.
From our results, there appears to be a strong link between CREB and improved memory performance in the mice that received rTMS. Overall, we saw elevated levels of CREB and its phosphorylation in both the hippocampus and FC. This result can be related to previous research which has also shown the ability of rTMS to increase CREB expression and its phosphorylation [20,21].
Increased pCREB is important for the transcriptional activity needed to convert short-term memory to a stable LTM [48e50] and blocking CREB prevents the consolidation of memory in the hours following the learning event [51]. Therefore, at the consolidation stage, it would be important if rTMS could increase pCREB. However, the role of the prolonged activation of CREB is less clear.
This study shows that phosphorylation of CREB was associated with improved memory and therefore may be important in the improved memory performance we saw in the rTMS-treated mice.
CREB activation can come from many synaptic pathways. Unfortunately, this study did not determine the exact pathway that would result in elevated pCREB. The phosphorylation of CAMKII was increased in both the hippocampus and FC which could cause the increased CREB phosphorylation [9e11].
As rTMS alters Ca2þ levels within neurons [18], this activation of CAMKII may have provided a direct pathway to activate CREB, additionally, CAMKII activation has been directly linked to GluR2 activity [52].
However, in the FC the SHAM mice also had elevated CAMKII but not CREB or GluR2. The synaptic staining revealed that the hippocampus plays a greater role in object recognition memory than the FC, so there may be other regulatory pathways involved that supported the activation of CREB from CAMKII after rTMS in the hippocampus and not the FC. From this study, it is difficult to distinguish whether the improvements from rTMS were solely due to the stimulation given 3 h post-encoding or the stimulations given within the 72-h.
Singular consolidating stimuli are effective in improving memory in human trials [3], however in rodent studies, consistent reactivation of memory pathways is essential for LTM maintenance [17].
Additionally, from the results in this study, we cannot identify whether the improved memory or neurobiochemical changes resulted from the rTMS stimulation during the consolidation phase improving the memory consolidation or if there was a secondary mechanism altering the retrieval of the memory independent of consolidation.
The results from this study show both short-term changes to the phosphorylation of proteins needed from synaptic consolidation and long-term changes to stable synaptic connections needed for memory maintenance. Therefore, it will be important for future studies to compare the effect of different stimulation time points and non-probe trial control groups to determine the exact mechanisms.
5. Conclusion
Together the results suggest that by promoting greater synaptic GluR2 and preventing its endocytosis, as well as maintaining synaptic connections in the PC and hippocampal CA1, rTMS can improve memory in the object recognition test.
We found evidence that these changes were possibly linked to CAMKII and CREB pathways in hippocampal neurons. These results further solidify delivering rTMS in the consolidation phase for LTM and altering synaptic plasticity, which could have greater implications in the clinical neuromodulation field, not only for improving memory but also in cases where greater retention of information is beneficial, for example, psychotherapy [53].
Future studies could employ similar methods to compare the consolidation and maintenance stimulation to directly determine the molecular mechanism contributing to the effect, as well as determine if this effect could be extended with prolonged maintenance rTMS.
CRediT authorship contribution statement
A.M. Heath: Conceptualization, Formal analysis, Investigation, Writing the original draft. M. Brewer: Investigation. J. Yesavage: Supervision, Writing e review & editing. M.W. McNerney: Supervision, Resources, Funding acquisition, Writing e review & editing.
Acknowledgments
The authors would like to thank Dr. Eugenia Poh for providing her figure for use in the manuscript. The authors would also like to the Ass. Prof. Jennifer Roger for providing the custom TMS coils used in this study.
This work was supported by the Department of Veteran's Affairs [grant number 5IK2BX004105-2]. Information in this document does not represent the views of the United States Government, the Department of Veteran's Affairs, or Stanford University School of Medicine.
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